1. Field of the Invention
The present invention relates to a method of increasing discrimination for a single base mismatch using a hurdle DNA and an artificially mismatched probe.
2. Description of the Related Art
A standard method for detecting a variation in a nucleic acid sequence depends upon specific recognition by one oligonucleotide strand of a complementary nucleic acid target strand. When the probe and the target are not identical, the affinity of the two strands for one another is reduced. Reduced affinity is manifested by a decrease in duplex thermal stability which can be conveniently monitored by measuring the duplex melting temperature (Tm). The difference in duplex melting temperatures (ΔTm) between, on the one hand, a perfectly matched probe and target and, on the other hand, the same probe with a second target that differs from the first target at one nucleotide, has proven useful in detecting sequence variations in DNA. Wallace, B. R. et al., Nucleic Acids Research 9:879 (1981) discriminated between short oligomers differing at a single base. Subsequently, Conner, B. J. et al., Proceedings of the National Academy of Sciences USA 80:278 (1983) used the Wallace approach to investigate point mutations in the β-globin gene. As a result, duplex thermal stability can be reasonably accurately predicted on the basis of sequences between probe and target.
Although hybridization can be a useful and powerful technique, it is limited in that the stability difference between a perfectly matched duplex and a mismatched duplex, particularly if the mismatch is only a single base, can be quite small, corresponding to a difference in Tm between the two of as little as 0.5° C. See Tibanyenda, N. et al., Eur. J. Biochem. 139:19 (1984) and Ebel, S. et al., Biochem. 31:12083 (1992). More importantly, it is understood that as the length of the oligomeric probe increases, the negative effect of a single base mismatch on overall duplex stability decreases. This is an important limitation because it is desirable to increase probe length to enhance hybridization specificity for single genes while excluding weakly related genes. Thus, the ability to specifically distinguish closely related genes has not kept pace with the desire to focus hybridization studies on increasingly narrow regions of the genome. What is desired is a method that improves the ability to distinguish closely related genes by increasing the difference in melting temperatures of duplexes formed between probe and target.
U.S. Pat. No. 6,764,693 discloses a nucleic acid hybridization method of improving an ability to distinguish a control nucleic acid target and a mutant nucleic acid target including sequence variation using a modified oligonucleotide. Artificial mismatch is induced using 1-(2′-deoxy-β-D-ribofuranosyl)-3-nitropyrrole as a base analogue to increase discrimination for a single base mismatch. However, synthesis of the base analogue is difficult and there is no description regarding blocking effect due to the addition of a hurdle DNA of the present invention.
The inventors of the present invention studied a method of increasing discrimination for a single base mismatch based on conventional technologies described above and found that by using the addition of a hurdle DNA and variation of a probe base, discrimination for a single base mismatch is increased. Thus, the present invention is completed.